EP3399307B1 - Peigne et cassette de gel d'électrophorèse - Google Patents

Peigne et cassette de gel d'électrophorèse Download PDF

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Publication number
EP3399307B1
EP3399307B1 EP18166992.0A EP18166992A EP3399307B1 EP 3399307 B1 EP3399307 B1 EP 3399307B1 EP 18166992 A EP18166992 A EP 18166992A EP 3399307 B1 EP3399307 B1 EP 3399307B1
Authority
EP
European Patent Office
Prior art keywords
gel
comb
cassette
tooth
well
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP18166992.0A
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German (de)
English (en)
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EP3399307A1 (fr
Inventor
Timothy Vale Updyke
Jennifer Miller
Thomas Diller
Siddarth Kannan
Robert Bennett
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
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Life Technologies Corp
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Publication of EP3399307A1 publication Critical patent/EP3399307A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • G01N27/44778—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis on a common gel carrier, i.e. 2D gel electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means

Definitions

  • the present invention relates to gel consumables.
  • Gel electrophoresis is a common procedure for the separation of biological molecules, such as DNA, RNA, polypeptides and proteins.
  • the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.
  • the basic unit used in this technique consists of a gel enclosed in a glass tube or sandwiched as a slab between glass or plastic plates.
  • Gels have an open molecular network structure, defining pores which are saturated with an electrically conductive buffered solution. These pores are large enough to admit passage of the migrating macromolecules through the gel.
  • the gel is placed in a chamber in contact with buffer solutions which make electrical contact between the gel and the cathode or anode of an electrical power supply.
  • a sample containing the macromolecules and a tracking dye is placed on top of the gel.
  • An electric potential is applied to the gel causing the sample macromolecules and tracking dye to migrate toward the bottom of the gel.
  • the electrophoresis is halted just before the tracking dye reaches the end of the gel. The locations of the bands of separated macromolecules are then determined.
  • the size of other macromolecules can be determined.
  • Polyacrylamide gels are commonly used for electrophoresis. Other gels suitable for electrophoresis include agarose gels and starch gels. Polyacrylamide gel electrophoresis or PAGE is popular because the gels are optically transparent, electrically neutral and can be made with a range of pore sizes.
  • Glass has typically been used to make molds for electrophoresis gels.
  • glass suffers from the disadvantage that it is fragile, difficult to form into particular shapes and expensive. It is easier and more economical to form gel molds from plastic materials by processes such as injection molding.
  • using plastic molds for casting electrophoresis gels may contribute to decreased resolution of the separated macromolecule bands. Decreased resolution of the macromolecule bands may be caused by macromolecules moving faster on the surface of the gel in contact with the mold than in the interior of the gel. This variation in migration rates between the surface of the gel and the interior lead may lead to smearing of the macromolecule bands.
  • One solution for increasing sample volume in a gel may be to increase the depth of the wells of a particular width and gel thickness. However, the increased volumes may produce increased sample heights above the gel, leading to thicker protein band starting zones, and lower resolution. If the second gel height is proportionately increased, then the resolving gel length will be proportionately decreased, also contributing to reduced resolution in a given gel cassette.
  • a gel cassette that would enable producing a gel of a particular thickness having a well capable of holding a sample volume that is at least double the well volume of the current methods, while maintaining the same sample height above the gel as a standard well of particular width and thickness.
  • US 5 284 565 A discloses a sample well insert with wedge-shaped profile for ultra-thin slab gels in electrophoresis.
  • WO 98/25136 A1 discloses an electrophoretic method and apparatus.
  • an apparatus for gel electrophoresis comprising a comb having wedge-shaped teeth configured to create a sample well in a 1 nun gel, wherein the sample well size is at least 10% larger than a gel having a sample well created by a standard comb.
  • an apparatus for gel electrophoresis comprising a comb having wedge-shaped teeth configured to create a sample well in a 1 mm gel, wherein the sample well size is at least 100% larger than a gel having a sample well created by a standard comb.
  • the sample well created by a standard mini gel ten teeth comb has a volume of 37.5 ⁇ L.
  • an apparatus for gel electrophoresis comprising a ten teeth mini gel comb having wedge-shaped teeth configured to create a sample well in a 1 mm gel, wherein the sample well has a volume of at least 70 ⁇ L.
  • an apparatus for gel electrophoresis comprising; a gel cassette having a lip extending at an angle from at least one exterior surface of the cassette; and a comb having at least one wedge-sbaped tooth.
  • an apparatus for gel electrophoresis comprising a gel cassette having a cavity; and a comb; and at least one tooth located on the comb, the at least one tooth having at least one side that is slanted, wherein the at least one tooth is located at least 0.335 inches from the edge of the cavity.
  • an apparatus for gel electrophoresis comprising a comb having wedge-shaped teeth configured to create sample wells configured to receive at least 10% more sample than a standard gel sample well.
  • the device may be configured to create a gel that isolates the desired component as a sharp band or a high resolution band or both.
  • an apparatus for gel electrophoresis comprising a gel cassette and a comb having at least one wedge-shaped tooth.
  • the gel cassette may include a retainer plate and a divider plate.
  • the plates may be formed by a process such as injection molding. Any suitable plastic may be used for forming the plates, such as for example purposes only polymers such as polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polystyrene, polyethylene, polymethyl polypropylene, and cellulose acetates or various co-polymers.
  • the plastic may be a transparent plastic so that the gel may be viewed inside the assembled cassette.
  • the cassette may be constructed from glass plates, or may be constructed from one glass plate and one plastic plate.
  • the device may be configured to create precast gels that lay flat on various surfaces. The ability to create precast gels that lay flat may be beneficial for product shipping and storage, as well as during routine handling by the user. Additionally, the gel cassette may lay flat in auto-loading machinery during gel manufacturing.
  • the divider plate has a series of features, bumps, or posts located along the length of either the sides of the plate, along the bottom length of the plate or both along the sides and bottom of the plate.
  • the posts may function as energy directors during the assembly process to create a uniform seal around the plate.
  • a slot may be located at the bottom portion of the divider plate, and in some embodiments, may or may not extend through the thickness of the plate. The slot may expose the gel inside the cassette to a buffer, such as an anode buffer, to complete or assist in completing an electrical path circuit during electrophoresis.
  • the retainer plate may have a spacer located on its interior surface.
  • the spacer may be any suitable shaped spacer, for example, a generally u-shaped raised spacer located on the interior surface.
  • the spacer may be located on the interior surface of one plate and contact the interior surface of the other plate.
  • a spacer may be located on the interior surface of both plates and the two spacers may contact each other when the cassette is formed. The spacer or spacers may further serve to help maintain the space between the divider plate and the retainer plate when the plates are joined to form a cassette.
  • a lip extends at some angle away from the exterior surface of one of the plates to form an opening.
  • Gel material may enter into the cassette through the opening.
  • a structure for forming sample wells in the gel may be introduced to the gel material through the opening.
  • the lip may extend from the exterior surface of one of the plates at an angle, for example at about a 70 degree angle from a plane perpendicular to the exterior surface of the plate, or 20 degrees from surface of the plate. In some embodiments, the angle may be between about 15 degrees and about 25 degrees, between about 10 degrees and 30 degrees, between about 5 degrees and 35 degrees, or any other suitable angle. In some embodiments, the lip may extend from the exterior surface of the plate by less than about 45 degrees.
  • the cassette has at least one interior surface which may be coated with a coating.
  • the coating may be au oxygen interference coating (a coating that forms an oxygen barrier that reduces diffusion from the surface of the plastic plates into the gel to prevent significant local variations in the rate of oxygen diffusion) such as, for example purposes only, a coating comprising SiO x , SiO. or SiO 2 .
  • the coating may be substantially transparent.
  • the interior surfaces maybe coated manually or mechanically and in some embodiments where two separate plates are formed into the cassette, the coating may be performed before or after joining the plates into the cassette.
  • an apparatus for gel electrophoresis comprising a comb having wedge-shaped teeth configured to create sample wells configured to receive at least 10% more sample than a standard gel sample well, increased sample size may allow for more sample to be loaded into the gel without causing damage to the gel and yet still produce sharp, high resolution bands. Additionally, a large sample well opening may facilitate loading samples with standard pipette tips instead of special drawn-out gel loading pipette tips.
  • the wedge-shaped wells permit a greater amount of sample to be loaded into a thinner gel (for example, approximately 1 mm thick gels).
  • the comb may be used with any of the cassettes described herein previously, or may be used with any other suitable cassette for creating a gel.
  • the wedge-shaped wells in the gel created by the wedge shaped teeth may be of a volume at least 10% greater than the volume of the wells created using a standard comb.
  • a standard comb may create wells having a volume of about 37.5 ⁇ L.
  • the wedge shaped wells may have a volume that is at least 20% greater, at least 50% greater, at least 100% greater, at least 150 % greater, at least 175% greater, at least 200% greater, at least 250% greater than a well created using a standard comb.
  • the sample well size created by the wedge well comb may be at least 41 ⁇ L.
  • the wedge-shaped wells may allow for loading an increased amount of sample while maintaining or improving band sharpness and resolution during electrophoresis while reducing edge effects.
  • the wedge shaped design of the wells formed by the teeth of a wedge-tooth comb allows more sample to be loaded into a well, while keeping the sample away flora the sides of the gel. Sample that is located toward the ends of the gel may not form as sharp bands or bands having high resolution. Positioning of the wells at such locations reduces the chance that the bands formed in the gel will turn up at the ends of the band or "smile" or turn.
  • the wedge-shaped wells created using the apparatus described herein have a greater cross sectional area at the top of the sample well as compared to the bottom of the sample well.
  • the increase in cross sectional area creates a lower voltage drop at any given constant voltage, current or wattage ut the top of the sample well compared to the bottom of the sample well.
  • the lower voltage drop, or field strength, in this area may lead to better sample stacking than in the thinner sections of the well and/or gel, or in gels with wells of the same thickness through out the sample loading volume.
  • the gel cassettes are about 10 cm by 10 cm for mini-gel cassettes.
  • the gels housed in the gel cassettes are about 7 cm in length (the running length) by about 8 cm wide.
  • the gel cassettes and gels run in the gel cassettes may be any suitable length.
  • the gel cassette width may be large enough to reduce edge effects in gels having at least 10 wells.
  • the assembled cassette may include a gap that runs around the outer perimeter of the cassette. The gap may facilitate the opening of the cassette for gel retrieval
  • the gel cassette may be opened with a gel knife or may be opened by hand.
  • the comb may be a ten-tooth comb, that is, a comb used for forming 10 sample wells in a gel.
  • the comb may be at least a single tooth comb, at least a 2-tooth comb, at least a five-tooth comb, at least a seven-tooth comb, at least a 9-tooth comb, at least a 10-tooth comb, at least a twelve-tooth comb, at least a 15-tooth comb, at least a 17-tooth comb, at least a 20-tooth comb, or any other comb having a suitable number of teeth.
  • the comb may be molded from any suitable plastic including, but not limited to polymers such as polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polystyrene, polyethylene, polymethyl polypropylene, cellulose acetates, co-polymers, polycarbonate, or any other suitable material.
  • the dimensions of the teeth may be uniform along the length of the comb or the teeth may have varying dimensions along the length of the comb.
  • the teeth may or may not be the same shape as the wedge shaped opening of the cassette.
  • the edge of the comb opposite to the teeth may serve as a handle that may facilitate the insertion and removal of the comb from the cassette.
  • the edge may be curved.
  • the comb may father include a back surface and a front surface.
  • the front and back surfaces are solid.
  • the front surface of the comb has a recessed space which facilitates gripping the comb by the user to facilitate the insertion of the comb into the cassette sis well as to remove the comb from the cassette once the gel and wells have been formed.
  • a divider plate 202 having an interior surface 206.
  • the divider plate has a series of features, bumps, or posts 240 located along the length of the sides of the plate 204 and along the bottom length of the plate.
  • the posts 240 may protrude from the divider plate 202 and upon attachment of the divider 202 to the retainer plate (204 in FIG. 2B ) the posts 240 may connect with the retainer plate.
  • the divider plate 202 may additionally include a ridge 241 that runs down the sides and across the bottom of the divider plate 202. The ridge may be used to seal the gel cavity on the assembled cassette.
  • a slot 214 may be located at the bottom portion of the divider plate 202, and in some embodiments, may or may not expend through the thickness of the plate 204.
  • the slot 214 may expose the gel inside the cassette to a buffer, such as an anode buffer, to complete or assist in completing an electrical path circuit during electrophoresis.
  • a groove 242 On the buck surface of the plate a groove 242 may or may not be present.
  • cut-outs 244, 246 may be located on the back surface 248 of the plate to facilitate removal of any sealant blocking the slot.
  • the top surface 252 may include a grip or a curved surface, as shown in FIG. 2A , to facilitate handling of the assembled cassette.
  • the retainer plate 304 and the divider plate 302 may be fabricated as a single piece of plastic. In some embodiments, the retainer plate 304 and the divider plate 302 may be two separate parts that are connected together. The two separate parts may be connected together by any suitable mechanism for attaching the plates together including, for example purposes only, glue, pressure fitting, welding, adhesive, clamps, clips, or any other suitable mechanism.
  • an electric circuit may be formed either through a conductive sealant material, or alternatively, the sealant can be removed from the exterior of the cassette, thereby exposing the gel inside the cassette to a buffer solution and thereby completing the electric circuit path.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Optical Measuring Cells (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Peptides Or Proteins (AREA)

Claims (14)

  1. Appareil pour électrophorèse sur gel comprenant :
    une cassette de gel (100) ; et
    un peigne (520) ayant au moins une dent (522), la dent ayant au moins un côté (533) qui est incliné et s'étendant d'un plan parallèle à une surface inférieure (534) de la dent à un angle compris entre 45 degrés et 90 degrés, la dent présentant un volume d'au moins 40 µL.
  2. Appareil selon la revendication 1, dans lequel la cassette de gel comporte une plaque de retenue (104) et une plaque de séparation (102).
  3. Appareil selon la revendication 1 ou 2, dans lequel la cassette de gel comporte une lèvre (216) s'étendant depuis la surface extérieure de la plaque de séparation et/ou de la plaque de retenue.
  4. Appareil selon la revendication 3, dans lequel la dent présente une surface plane à l'extrémité de la dent.
  5.  
  6. Appareil selon la revendication 0, dans lequel la cassette présente au moins une surface intérieure.
  7. Appareil selon la revendication 6, dans lequel l'au moins une surface intérieure est revêtue d'un revêtement.
  8. Appareil selon la revendication 7, dans lequel le revêtement est un revêtement d'interférence avec l'oxygène.
  9. Appareil selon la revendication 8, dans lequel le revêtement d'interférence avec l'oxygène est choisi parmi SiO et/ou SiO2.
  10. Appareil selon la revendication 1, dans lequel la dent présente un volume de : au moins 41 µL, au moins environ 45 µL, au moins environ 55 µL, au moins 60 µL, au moins environ 70 µL, au moins environ 75 µL, au moins environ 90 µL, au moins environ 100 µL, au moins environ 110 µL ou au moins environ 130 µL.
  11. Appareil selon la revendication 1 :
    dans lequel le peigne est un peigne à un puits, un peigne à neuf puits, un peigne à dix puits, un peigne à douze puits, un peigne à quinze puits ou un peigne à dix-sept puits.
  12. Appareil selon la revendication 1, comprenant une zone évidée sur l'arrière du peigne.
  13. Appareil selon la revendication 12, dans lequel la zone évidée est conçue pour empêcher la formation d'une peau pendant la formation du gel.
  14. Appareil selon la revendication 1, dans lequel l'au moins une dent est située à au moins 0,335 pouce du bord de la cavité.
EP18166992.0A 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse Active EP3399307B1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US23629309P 2009-08-24 2009-08-24
US23679509P 2009-08-25 2009-08-25
US23728709P 2009-08-26 2009-08-26
US23719509P 2009-08-26 2009-08-26
EP14195596.3A EP2853891B1 (fr) 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse
PCT/US2010/046506 WO2011028532A1 (fr) 2009-08-24 2010-08-24 Cassette et peigne d'électrophorèse sur gel
EP16206945.4A EP3168610B1 (fr) 2009-08-24 2010-08-24 Appareil gel-électrophorese avec cassette et comb
EP10814246.4A EP2470691B1 (fr) 2009-08-24 2010-08-24 Cassette et peigne d'électrophorèse sur gel

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
EP14195596.3A Division EP2853891B1 (fr) 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse
EP10814246.4A Division EP2470691B1 (fr) 2009-08-24 2010-08-24 Cassette et peigne d'électrophorèse sur gel
EP16206945.4A Division EP3168610B1 (fr) 2009-08-24 2010-08-24 Appareil gel-électrophorese avec cassette et comb

Publications (2)

Publication Number Publication Date
EP3399307A1 EP3399307A1 (fr) 2018-11-07
EP3399307B1 true EP3399307B1 (fr) 2020-11-11

Family

ID=43604426

Family Applications (5)

Application Number Title Priority Date Filing Date
EP18166992.0A Active EP3399307B1 (fr) 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse
EP16206945.4A Active EP3168610B1 (fr) 2009-08-24 2010-08-24 Appareil gel-électrophorese avec cassette et comb
EP10814246.4A Active EP2470691B1 (fr) 2009-08-24 2010-08-24 Cassette et peigne d'électrophorèse sur gel
EP14195596.3A Active EP2853891B1 (fr) 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse
EP10812553.5A Active EP2470891B1 (fr) 2009-08-24 2010-08-24 Electrophorèse sur gel, imagerie et procédés, dispositifs, systèmes et matériaux d'analyse

Family Applications After (4)

Application Number Title Priority Date Filing Date
EP16206945.4A Active EP3168610B1 (fr) 2009-08-24 2010-08-24 Appareil gel-électrophorese avec cassette et comb
EP10814246.4A Active EP2470691B1 (fr) 2009-08-24 2010-08-24 Cassette et peigne d'électrophorèse sur gel
EP14195596.3A Active EP2853891B1 (fr) 2009-08-24 2010-08-24 Peigne et cassette de gel d'électrophorèse
EP10812553.5A Active EP2470891B1 (fr) 2009-08-24 2010-08-24 Electrophorèse sur gel, imagerie et procédés, dispositifs, systèmes et matériaux d'analyse

Country Status (6)

Country Link
US (8) US20110042213A1 (fr)
EP (5) EP3399307B1 (fr)
JP (4) JP2013502598A (fr)
CN (2) CN102656448A (fr)
IN (2) IN2012DN01871A (fr)
WO (2) WO2011028532A1 (fr)

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US20140231260A1 (en) 2014-08-21
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US20200278317A1 (en) 2020-09-03
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